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1.
Nature ; 587(7835): 657-662, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32726803

RESUMO

The papain-like protease PLpro is an essential coronavirus enzyme that is required for processing viral polyproteins to generate a functional replicase complex and enable viral spread1,2. PLpro is also implicated in cleaving proteinaceous post-translational modifications on host proteins as an evasion mechanism against host antiviral immune responses3-5. Here we perform biochemical, structural and functional characterization of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PLpro (SCoV2-PLpro) and outline differences with SARS-CoV PLpro (SCoV-PLpro) in regulation of host interferon and NF-κB pathways. SCoV2-PLpro and SCoV-PLpro share 83% sequence identity but exhibit different host substrate preferences; SCoV2-PLpro preferentially cleaves the ubiquitin-like interferon-stimulated gene 15 protein (ISG15), whereas SCoV-PLpro predominantly targets ubiquitin chains. The crystal structure of SCoV2-PLpro in complex with ISG15 reveals distinctive interactions with the amino-terminal ubiquitin-like domain of ISG15, highlighting the high affinity and specificity of these interactions. Furthermore, upon infection, SCoV2-PLpro contributes to the cleavage of ISG15 from interferon responsive factor 3 (IRF3) and attenuates type I interferon responses. Notably, inhibition of SCoV2-PLpro with GRL-0617 impairs the virus-induced cytopathogenic effect, maintains the antiviral interferon pathway and reduces viral replication in infected cells. These results highlight a potential dual therapeutic strategy in which targeting of SCoV2-PLpro can suppress SARS-CoV-2 infection and promote antiviral immunity.


Assuntos
COVID-19/imunologia , COVID-19/virologia , Proteases Semelhantes à Papaína de Coronavírus/química , Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Imunidade Inata , SARS-CoV-2/enzimologia , SARS-CoV-2/imunologia , Animais , Proteases Semelhantes à Papaína de Coronavírus/antagonistas & inibidores , Citocinas/química , Citocinas/metabolismo , Enzimas Desubiquitinantes/antagonistas & inibidores , Enzimas Desubiquitinantes/química , Enzimas Desubiquitinantes/metabolismo , Humanos , Fator Regulador 3 de Interferon/metabolismo , Interferons/imunologia , Interferons/metabolismo , Camundongos , Modelos Moleculares , Simulação de Dinâmica Molecular , NF-kappa B/imunologia , NF-kappa B/metabolismo , Ligação Proteica , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Ubiquitinação , Ubiquitinas/química , Ubiquitinas/metabolismo , Tratamento Farmacológico da COVID-19
2.
J Biol Chem ; 297(2): 100925, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34214498

RESUMO

Apart from prevention using vaccinations, the management options for COVID-19 remain limited. In retrospective cohort studies, use of famotidine, a specific oral H2 receptor antagonist (antihistamine), has been associated with reduced risk of intubation and death in patients hospitalized with COVID-19. In a case series, nonhospitalized patients with COVID-19 experienced rapid symptom resolution after taking famotidine, but the molecular basis of these observations remains elusive. Here we show using biochemical, cellular, and functional assays that famotidine has no effect on viral replication or viral protease activity. However, famotidine can affect histamine-induced signaling processes in infected Caco2 cells. Specifically, famotidine treatment inhibits histamine-induced expression of Toll-like receptor 3 (TLR3) in SARS-CoV-2 infected cells and can reduce TLR3-dependent signaling processes that culminate in activation of IRF3 and the NF-κB pathway, subsequently controlling antiviral and inflammatory responses. SARS-CoV-2-infected cells treated with famotidine demonstrate reduced expression levels of the inflammatory mediators CCL-2 and IL6, drivers of the cytokine release syndrome that precipitates poor outcome for patients with COVID-19. Given that pharmacokinetic studies indicate that famotidine can reach concentrations in blood that suffice to antagonize histamine H2 receptors expressed in mast cells, neutrophils, and eosinophils, these observations explain how famotidine may contribute to the reduced histamine-induced inflammation and cytokine release, thereby improving the outcome for patients with COVID-19.


Assuntos
Famotidina/farmacologia , Antagonistas dos Receptores Histamínicos/farmacologia , SARS-CoV-2/efeitos dos fármacos , Receptor 3 Toll-Like/metabolismo , Células A549 , Sítios de Ligação , Células CACO-2 , Quimiocina CCL2/metabolismo , Proteases 3C de Coronavírus/metabolismo , Células HeLa , Humanos , Fator Regulador 3 de Interferon/metabolismo , Interleucina-6/metabolismo , Simulação de Acoplamento Molecular , NF-kappa B/metabolismo , Ligação Proteica , SARS-CoV-2/fisiologia , Transdução de Sinais , Receptor 3 Toll-Like/química , Replicação Viral
3.
FASEB J ; 33(2): 1927-1945, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30230921

RESUMO

The mechanism by which the endoplasmic reticulum (ER) ubiquitin ligases sense stress to potentiate their activity is poorly understood. GP78, an ER E3 ligase, is best known for its role in ER-associated protein degradation, although its activity is also linked to mitophagy, ER-mitochondria junctions, and MAPK signaling, thus highlighting the importance of understanding its regulation. In healthy cells, Mahogunin really interesting new gene (RING) finger 1 (MGRN1) interacts with GP78 and proteasomally degrades it to alleviate mitophagy. Here, we identify calmodulin (CaM) as the adapter protein that senses fluctuating cytosolic Ca2+ levels and modulates the Ca2+-dependent MGRN1-GP78 interactions. When stress elevates cytosolic Ca2+ levels in cultured and primary neuronal cells, CaM binds to both E3 ligases and inhibits their interaction. Molecular docking, simulation, and biophysical studies show that CaM interacts with both proteins with different affinities and binding modes. The physiological impact of this interaction switch manifests in the regulation of ER-associated protein degradation, ER-mitochondria junctions, and relative distribution of smooth ER and rough ER.-Mukherjee, R., Bhattacharya, A., Sau, A., Basu, S., Chakrabarti, S., Chakrabarti, O. Calmodulin regulates MGRN1-GP78 interaction mediated ubiquitin proteasomal degradation system.


Assuntos
Calmodulina/metabolismo , Neurônios/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Receptores do Fator Autócrino de Motilidade/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Animais , Sinalização do Cálcio , Calmodulina/química , Calmodulina/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Células HeLa , Humanos , Camundongos , Simulação de Acoplamento Molecular , Neurônios/citologia , Complexo de Endopeptidases do Proteassoma/genética , Receptores do Fator Autócrino de Motilidade/química , Receptores do Fator Autócrino de Motilidade/genética , Ubiquitina/genética , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética
4.
Autophagy ; 2024 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-39291751

RESUMO

The KEAP1-NFE2L2 axis is essential for the cellular response against metabolic and oxidative stress. KEAP1 is an adaptor protein of CUL3 (cullin 3) ubiquitin ligase that controls the cellular levels of NFE2L2, a critical transcription factor of several cytoprotective genes. Oxidative stress, defective autophagy and pathogenic infections activate NFE2L2 signaling through phosphorylation of the autophagy receptor protein SQSTM1, which competes with NFE2L2 for binding to KEAP1. Here we show that phosphoribosyl-linked serine ubiquitination of SQSTM1 catalyzed by SidE effectors of Legionella pneumophila controls NFE2L2 signaling and cell metabolism upon Legionella infection. Serine ubiquitination of SQSTM1 sterically blocks its binding to KEAP1, resulting in NFE2L2 ubiquitination and degradation. This reduces NFE2L2-dependent antioxidant synthesis in the early phase of infection. Levels of serine ubiquitinated SQSTM1 diminish in the later stage of infection allowing the expression of NFE2L2-target genes; causing a differential regulation of the host metabolome and proteome in a NFE2L2-dependent manner.

5.
Nat Commun ; 15(1): 7939, 2024 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-39261458

RESUMO

Acinetobacter baumannii is a pathogenic and multidrug-resistant Gram-negative bacterium that causes severe nosocomial infections. To better understand the mechanism of pathogenesis, we compare the proteomes of uninfected and infected human cells, revealing that transcription factor FOS is the host protein most strongly induced by A. baumannii infection. Pharmacological inhibition of FOS reduces the cytotoxicity of A. baumannii in cell-based models, and similar results are also observed in a mouse infection model. A. baumannii outer membrane vesicles (OMVs) are shown to activate the aryl hydrocarbon receptor (AHR) of host cells by inducing the host enzyme tryptophan-2,3-dioxygenase (TDO), producing the ligand kynurenine, which binds AHR. Following ligand binding, AHR is a direct transcriptional activator of the FOS gene. We propose that A. baumannii infection impacts the host tryptophan metabolism and promotes AHR- and FOS-mediated cytotoxicity of infected cells.


Assuntos
Infecções por Acinetobacter , Acinetobacter baumannii , Cinurenina , Receptores de Hidrocarboneto Arílico , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores de Hidrocarboneto Arílico/genética , Acinetobacter baumannii/metabolismo , Acinetobacter baumannii/genética , Acinetobacter baumannii/efeitos dos fármacos , Humanos , Animais , Camundongos , Infecções por Acinetobacter/microbiologia , Infecções por Acinetobacter/metabolismo , Cinurenina/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Triptofano/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Interações Hospedeiro-Patógeno
6.
Nat Cell Biol ; 25(5): 685-698, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-37024685

RESUMO

Acute lysosomal membrane damage reduces the cellular population of functional lysosomes. However, these damaged lysosomes have a remarkable recovery potential independent of lysosomal biogenesis and remain unaffected in cells depleted in TFEB and TFE3. We combined proximity-labelling-based proteomics, biochemistry and high-resolution microscopy to unravel a lysosomal membrane regeneration pathway that depends on ATG8, the lysosomal membrane protein LIMP2, the RAB7 GTPase-activating protein TBC1D15 and proteins required for autophagic lysosomal reformation, including dynamin-2, kinesin-5B and clathrin. Following lysosomal damage, LIMP2 acts as a lysophagy receptor to bind ATG8, which in turn recruits TBC1D15 to damaged membranes. TBC1D15 interacts with ATG8 proteins on damaged lysosomes and provides a scaffold to assemble and stabilize the autophagic lysosomal reformation machinery. This potentiates the formation of lysosomal tubules and subsequent dynamin-2-dependent scission. TBC1D15-mediated lysosome regeneration was also observed in a cell culture model of oxalate nephropathy.


Assuntos
Autofagia , Dinamina II , Dinamina II/metabolismo , Membranas Intracelulares/metabolismo , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Lisossomos/metabolismo
7.
Elife ; 92020 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-33185526

RESUMO

Legionella pneumophila causes a severe pneumonia known as Legionnaires' disease. During the infection, Legionella injects more than 300 effector proteins into host cells. Among them are enzymes involved in altering the host-ubiquitination system. Here, we identified two LegionellaOTU (ovarian tumor)-like deubiquitinases (LOT-DUBs; LotB [Lpg1621/Ceg23] and LotC [Lpg2529]). The crystal structure of the LotC catalytic core (LotC14-310) was determined at 2.4 Å. Unlike the classical OTU-family, the LOT-family shows an extended helical lobe between the Cys-loop and the variable loop, which defines them as a unique class of OTU-DUBs. LotB has an additional ubiquitin-binding site (S1'), which enables the specific cleavage of Lys63-linked polyubiquitin chains. By contrast, LotC only contains the S1 site and cleaves different species of ubiquitin chains. MS analysis of LotB and LotC identified different categories of host-interacting proteins and substrates. Together, our results provide new structural insights into bacterial OTU-DUBs and indicate distinct roles in host-pathogen interactions.


Assuntos
Bactérias/enzimologia , Enzimas Desubiquitinantes/metabolismo , Linhagem Celular , Enzimas Desubiquitinantes/genética , Escherichia coli , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Humanos , Legionella , Legionelose , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Ubiquitinação
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